The present invention is in the field of genetic analysis for medical diagnosis, genetic variation research, or genetic engineering. More specifically, the present invention is in the field of nucleic acid amplification.
Many differences in living organisms, including biological traits, characteristics or disease susceptibilities, are closely related to their genetic variations. Therefore, it is desirable to understand genetic variations of organisms so that useful information can be obtained to help select organisms with desirable traits or characteristics or predict an organism""s disease susceptibility and thus provide proper treatments.
Most often, the study of genetic variations, for example the study of genetic polymorphism, involves the analysis of nucleic acid sequences in DNA or RNA. The sequences of interest may be low in occurrence in nucleic acid samples. On the other hand, undesirable sequences may have high occurrence in samples. Some of these undesirable sequences are repetitive sequences. The high occurrence of unwanted sequences may cause serious interference when analyzing genetic variations because they can produce a significant background noise in genetic detection. The problem becomes more severe when an amplification process is employed to increase the copy numbers of the sequences of interest because the amplification process may amplify both interested sequences and unwanted sequences indiscriminately. The present invention is directed to decreasing the possibility of amplifying unwanted sequences during an amplification process so that sequences of interest can be amplified while unwanted sequences will not be amplified, thus decreasing the background noise in genetic variation analysis. The present invention is especially useful to suppress the amplification of repetitive sequences.
According to the present invention, methods are provided to block unwanted nucleic acid sequences from being amplified in a nucleic acid amplification process by adding blocking molecules that bind to the undesirable nucleic acids sequences and thus preventing the amplification of undesired sequences in the process. The methods can be used to block any undesirable sequences and are especially useful for blocking repetitive sequences.
In one embodiment of the invention, a method for blocking amplification of undesirable DNA sequences during a DNA amplification process comprises the following steps: providing DNA samples from cells or homogenized tissues; fragmenting the DNA by restriction enzymes or DNase followed by end modification and adapter ligation; blocking undesirable DNA sequences by peptide nucleic acids having complimentary sequences to the undesirable DNA sequences prior to or during amplifying the DNA samples by polymerase chain reaction (PCR) with proper reagents, enzymes and primers.
In another embodiment of the invention, a kit is constructed to carry out the blocking method. The kit comprises Cot-1 cRNA sequences as the blocking molecules, a restriction digestion enzyme, an adapter comprising a primer sequence and a cohesive end corresponding to the restriction site specified by the restriction enzyme, a ligase, and corresponding primers.